KR100485686B1 - Method for production of oligosaccharide from Laminaria japonica - Google Patents

Abstract

The present invention relates to a method for preparing a true damashima-derived oligosaccharide, comprising the culture supernatant of an Escherichia coli strain inserted with pLK108S (KFCC-11303) or endo-beta-1,3-glu recovered from the same. It is very useful for the food and pharmaceutical industry because it is possible to provide a method for producing oligosaccharides in high efficiency in a short time by adding kinase to the laminarin polysaccharide fraction purified from Chada Shima from enzymatic reaction.

Description

Method for producing oligosaccharide derived from Chadashima {Method for production of oligosaccharide from Laminaria japonica}

The present invention relates to a method for preparing a true dashima derived oligosaccharide, and more particularly, Escherichia coli ( E ) into which a recombinant vector pLK108S (KFCC-11303) containing an endo-beta-1,3-glucanase gene is inserted. coli ) A method for producing oligosaccharides from Chedamasima using an enzyme produced by a DH5α transformant.

There are many glycans in nature that we can use industrially. Among them, a combination of beta-1,3-glucan, La minariah japonica main structure and the storage polysaccharide laminarin (laminarin), such as (Laminaria japonica) (β-1,3 -glucan) is present in large amounts in brown algae ocean Known. Laminarin is an unbranched β-1,3-glucan polymer consisting of glucose residues. The structure is derived from Laminaria digitata , a representative brown algae, and has two types. There are M type and G type, and they exist in the ratio of 3: 1. Type M has been reported to form about 20-30 glucose residues linked to mannitol residues and type G contains about 22-28 glucose residues (Steve, MR, Graeme C., Antony). , B. 1996. Analysis of the structural heterogeneity of laminarin by electrospray-ionisation-mass spectrometry Carbohydr.Res . 28: 187-201). In addition, beta-1,3-glucan is well known as a cell wall component of fungi and yeast, and Saccharomyces cerevisiae and Saccharomyces cerevisiae Debaryomyces hansenii , Hansenula ciferrii , Candida albicans , Pyricularia oryzae , Aspergillus oryzae , Neurospo Molecular cloning of a cell wall exo-β-1,3-glucanase from Saccharomyces cerevisiae has been reported as a cell wall component of a variety of yeasts and fungi, such as la la crasa ( Neurospora crassa ) (Klebl, F. and Tanner, W. 1989. J. Bacteriol. 171: 6529, Hilary, J., Donald J. and Patick, A. 1999. Hydrolase and transferase activities of the β-1,3-exoglucanase of Candida albicans Eur. J. Biochem. 263: 889-895, Salazar, O., Molitor J, and Asenjo, JA 1999. Cloning and expression of an Oerskovia xanthineolytica β-1,3-glucanase in Escherichia coli Biotechnol. Lett. 21: 797-802.

Beta-glucan is well known as a biological response modifier (BRM) that is widely present in nature and has recently been used as a plant and medicine (Naohito Ohno et al. 2000, Antitumor 1,3-b-glucan from cultures fruit body of Sparossis crispa, Biol.Pharm Bull , 23 (7): 866-872). Laminarin, known as beta-1,3-glucan, has been reported to have been isolated and purified from Eisenia bicyclis , a type of brown algae (Masaakira Maeda and Kazutosi Nisizawa 1968, Fine structure of Laminaran of Eisenia bicyclis, J. Biochem. 63 (2): 199-206), and also Ljudmila A, et al., obtained from various far-eastern brown seaweeds. We have systematically reported biochemical characteristics such as molecular weight of naryn and the structure of residue ends (Ljudmila A. Elyakova and Tatiana N. Zvyagintseva 1974, A study of the laminarins of some far-eastern, brown seaweeds Carbohydr. Res. 34: 241-248). In addition, water-soluble polysaccharides extracted from far-east brown seaweeds have been reported anti-complementary bioactivity (Zvyagintseva TN et al. 2000, Inhibition of complement activation by water-soluble polysaccharides of some far-eastern brown seaweeds, Comp. Physiol.C Toxicol Pharmacol 126 (3): 209-215), beta-1,3-glucan isolated from mushrooms has been reported as an anticancer active substance (Naohito Ohno et al. 2000, Antitumor 1,3-b-glucan from cultures fruit body of Sparossis crispa, Biol.Pharm Bull , 23 (7): 866-872, Hisami Shinohara et al. 1988, Antitumor activity and structural characterization of a 1,3-b-glucan extracted with cold alkali from sclerotia of Sclerotinia sclerotiorum IFO 9395 , Chem. Phrm. Bull. 36 (2): 819-823, Naohito Ohno et al. 1986, Structure and antitumor activity of a b-1,3-glucan isolated from the culture filtrate of Sclerotinia sclerotiorum IFO 9395 , Chem. Pharm. Bull ., 34 (3): 1362-1365, Hirohide Mimura et al. 1985, Purification, antitumor activity, and structural characterization of b-1,3-glucan from Peziza vesiculosa , Chem. Pharm. Bull . 33 (11): 5096-5099, Naohito Ohno et al. 1984, Antitumor activity and structural characterization of glucans extracted from cultured fruit bodies of Grifola frondosa, Chem. Pharm. Bull. 32 (3): 1142-1151).

Currently, there have been some reports on the separation and purification of fucoidan from kelp (Koo Jae-geun, Gil-seok Cho, Do-ryong Rong, Woo Soon-ja. Extraction and purification of Fucoidan from Korean kelp and seaweed. 227-236) Laminar studies have not been reported.

In this regard, the present inventors have identified E. coli ( E. coli ). (pLK108S) To provide a new oligosaccharide production method from laminarin polysaccharides isolated and purified from Korean Chadashima using the culture supernatant of transformants or endo-beta-1,3-glucanase recovered therefrom.

Accordingly, an object of the present invention is to obtain a culture supernatant of an Escherichia coli strain into which recombinant pLK108S (KFCC-11303) is inserted or endo-beta-1,3-glucanase recovered therefrom. The present invention provides a method for producing an oligosaccharide derived from true kamashima in a short time by enzymatic reaction by addition to the separated laminarin polysaccharide fraction.

The object of the present invention is to isolate the culture supernatant of E. coli strains into which recombinant pLK108S (KFCC-11303) is inserted or endo-beta-1,3-glucanase recovered therefrom from Chamdashima. The present invention has been completed by discovering that it is possible to efficiently prepare a true kadashima-derived oligosaccharide in a short time by adding the purified laminarin polysaccharide fraction to enzymatic reaction.

Hereinafter, the configuration of the present invention.

The present invention comprises the steps of extracting the Charmedima laminarin fraction; Preparing an endo-beta-1,3-glucanase enzyme solution and subjecting the enzyme solution to an enzyme reaction by adding a laminarin fraction derived from true shimashima obtained in the step; Analyzing the hydrolysis products obtained in the above step using TLC and HPLC to identify oligosaccharides; And retrieving the formal activity of the oligosaccharides derived from true kamashima of the above step.

In order to achieve the above object, the present invention refers to a culture supernatant of an Escherichia coli strain into which pLK108S (KFCC-11303) is inserted or an endo-beta-1,3-glucanase recovered therefrom. Provided is a method for producing oligosaccharide derived from Chadashima, characterized in that oligosaccharides are prepared by adding to the laminarine polysaccharide fraction purified from the above.

Hereinafter, the present invention will be described in more detail.

The present invention is a supernatant of Escherichia coli strain into which pLK108S (KFCC-11303) is inserted, or laminarine, isolated and purified from endo-beta-1,3-glucanase recovered from Chadashima. The present invention relates to a method for producing true polysaccharide derived oligosaccharides by addition to a polysaccharide fraction and enzymatic reaction.

The plasmid pLK108S (KFCC-11303) used in the present invention is a recombinant plasmid pLK432 prepared by subcloning β-1,3-glucanase DNA fragment and pUB110 having kanamycin resistance to be expressed in Bacillus cells. After isolation and purification and cleavage with restriction enzymes, the two DNAs are linked to each other.

The deposited pLK108S (KFCC-11303) was introduced into E. coli by E. coli in a conventional genetic method, transformed and cultured to directly recover the culture supernatant, or endo-beta-1,3- Glucanase is extracted.

The endo-beta-1,3-glucanase is added to the laminarin polysaccharide fraction separated and purified from Chadashima, and subjected to enzymatic reaction to prepare oligosaccharides from Chadashima. The oligosaccharides are mainly laminaribiose (G2) and some are laminaripentaose (G5).

At this time, preferably, the true shimashima derived laminarin is a step of extracting hot water dried kelp; Centrifuging for 20 minutes at 5000 rpm when precipitation occurs by adding 4 times the volume of ethanol to the extract (CLP); Performing ultrafiltration of the crude hydrothermal extract from the above step using a 0.1-m ² membrane (LP); Filtering the solution obtained in the above step using a 100 kD membrane to remove the polymer; Concentrating the solution obtained in the above step using a 1 kD membrane and then lyophilizing to remove the salt of true kelp; Concentrating the crude polysaccharide 10-fold using hot water extraction and minitan-II and then gel filtration (LPI); And measuring the total equivalent weight of the CLP, LP and LP1 fractions of the above step through a sulfuric acid-phenol assay and purifying the fraction containing laminarine polysaccharides. This is a condition in which laminarin can be efficiently extracted from chamdashima.

As described above, endo-beta-1,3-glucanase expressed from E. coli is faster than endo-beta-1,3-glucanase expressed from other strains, and also High efficiency and excellent effect.

Hereinafter, the present invention will be described in more detail with reference to the following Examples and Experimental Examples. However, the following Examples and Experimental Examples are only for illustrating the present invention, the scope of the present invention is not limited to these.

Example 1 Preparation of Recombinant Shuttle Plasmid pLK108S (KFCC-11303)

From Bacillus Circus IJ2001 Recombinant plasmid pLK432 was constructed by subcloning again except for 0.8 kb of the β-1,3-glucannase 4.4 kb DNA fragment derived from the β-1,3-glucannase gene. PUB110 (4.5 kb Km ^r ) derived from Bacillus subtilis RM125 and a recombinant plasmid pLK432 (4.6 kb, Ap ^r ) containing β-1,3-glucanase gene from E. coli for the production of shuttle plasmids. Were separated and purified, respectively, and digested with restriction enzyme Xba I, followed by ligation of the two DNAs. Analysis and confirmation of the linked recombinant shuttle plasmid was performed through agarose gel (0.7-1.5) electrophoresis of DNA fragments cut with restriction enzymes. This recombinant shuttle plasmid pLK108S was deposited on April 10, 2002 with the deposit number KFCC 11303 at the Korea Microorganism Conservation Center.

Example 2 Simple Extraction of Chadashima Laminarin Fraction

1 kg of dried kelp was extracted with hot and pressurized water for 3 hours. After extracting the crude extract (crude extract) with 4 times the volume of ethanol was centrifuged for 20 minutes at 5000rpm at 4 ℃ (CLP). This was dissolved in sterile distilled water and dried 1mL to measure the extraction yield. Aqueous crude extract extracted from hot water was subjected to ultrafiltration using 0.1-m ² membranes (LP). To remove the polymer was filtered using a 100 kD membrane (membrane), concentrated to use a 1 kD membrane (membrane) to remove the salt of true kelp, and then lyophilized. Crude polysaccharide obtained by hot water extraction and Minitan-II was concentrated 10-fold, and then gel-filtered using Sephadex G-50 (Sephadex G-50) packed in an XK26 column (LP1). . The total equivalents of each fraction were determined via a sulfuric acid-phenol assay and the fractions containing laminarin polysaccharides were purified.

The laminarin polysaccharide fraction was extracted by hot water extraction, ultrafiltration and gel filtration and named LP1 fraction (FIG. 1). The yield of the extracted LP1 fraction was 1-2%. The molecular weight of the laminarin polysaccharide fraction extracted from Chedamashima was made by using a standard polysaccharide and analyzed using it. As a result, the range of the average molecular weight was found to be 5-10 kDa (FIGS. 2 and 3).

Example 3 Endo-beta-1,3-glucanase Enzyme Reaction

The enzyme solution was prepared and reacted as follows.

In order to prepare the enzyme solution S. serie cyano kolriyi (E. coli) were used to DH5a (pLK108S), LB liquid culture using the medium at 37 ℃ shaken for 16 hours after separation 10 minutes and centrifuged at 12,000rpm to pellet remaining real tumefaciens (bacterial pellet) was suspended in 0.05M citric acid buffer (pH 5.4), and ultrasonically crushed at 20 ㎑ for 3 minutes, and then the supernatant obtained by centrifugation at 12,000 rpm for 10 minutes was used for the enzymatic reaction. 0.05M citric acid buffer (pH5.4) to which fraction (1%) was added was used. Endo-beta-1,3-glucanase reaction was determined by unit of enzyme and 1mL of substrate was mixed and reacted at 55 ° C. at time. Reducing sugar was measured by colorimetric measurement at 550 nm according to the method by 3,5-dinitrosalicylic acid (DNS), and 1 unit of the amount of enzyme that produces 1 μmol of reducing sugar at 55 ° C for 1 minute. Marked as.

After reacting the endo-beta-1,3-glucanase enzyme with the laminarin polysaccharide fraction extracted from Chadashima, the degradation product was confirmed and showed a new hydrolysis pattern.

Example 4 Analysis of Hydrolysis Products of the Laminarin Polysaccharide Fraction

The hydrolysis products of the laminarin polysaccharide fractions were analyzed using TLC and HPLC.

First, TLC was performed by loading 5 μl of silica gel 60 TLC plate after completion of the time-phase reaction of endo-beta-1,3-glucanase. At this time, isoamylalchohol: ethanol: ammonia: water (50: 60: 1: 30) was used as a developing solvent, and then completely dried in air, 5% methoxybenzaldehyde and 5% sulfuric acid, After spraying with a solution containing a small amount of glacial acetate and 90% ethanol, the reaction was carried out at 120 ° C. for 15 minutes. TLC analysis showed some glucose and laminaribiose (G2) as main products (FIG. 4).

HPLC (Agilent 1100 series) analysis was performed to confirm the molecular weight of the extracted laminarin polysaccharide fraction and the hydrolyzate after enzymatic reaction. The GPC column was used at 40 degreeC for the confirmation of molecular weight. At this time, the mobile phase using distilled water, and analyzed by flowing at a flow rate of 1mL / min. Carbohydrate NH ₂ column (carbohydrate NH ₂ column) was used to confirm the decomposition products at 40 ℃, the mobile phase was used 70% acetonitrile (CH ₃ CN), flowing at a flow rate of 0.9mL / min Analyzed. As a result, a little laminaripentaose (G5) and glucose (G1) including laminaribioz (G2) were identified similarly to TLC analysis (FIG. 5).

Example 5. Formal Activity Screening of True Komashima Oligosaccharides

Bifidobacterium adolescentis KCCM 11206, Bifidobacterium infantis KCCM 11027, Lactobacillus acidophilus KCTC 3179, Lactobacillus cassis casei ) KCTC 1046, Lactobacillus fermentum KCCM 40401 and Lactobacillus reuteri KCCM 40717 was used. Bifidobacterium (Bifidobacterium) is RC (reinforced clostridial) [3g yeast extract, beef extract (Beef extract) 10g, peptone 10g, soluble starch 1g, glucose 5g, cysteine hydrochloride (cysteine hydrochloride) 0.5g, NaCl 5g , sodium acetate 3g / L] medium was used as a base, Lactobacillus ( Mactobacillus ) MRS [peptone 10g, beef extract 10g, yeast extract 5g, dextrose 20g, Tween 80 1g, ammonium citrate 5g, 0.1 g of magnesium sulfate, 0.05 manganese sulfate, dipotassium phosphate 2 g / 1 L] medium was used as a base. In the above basic medium, except for the glucose component, carbon source free medium was used, and the medium to which oligosaccharides generated by endo-beta-1,3-glucanase enzyme reaction was added. The growth rate of the indicator strain was anaerobically cultured at 37 ° C. and measured at 665 nm absorbance, and acid production was measured with a pH meter. Lastly, the consumption of oligosaccharides of indicator strains was analyzed by TLC.

On the other hand, the functional activity was searched using the true polysaccharide oligosaccharides, and the results showed that the oligosaccharides were used in both Bifidobacterium and Lactobacillus strains. First, as a result of checking the growth rate, the growth rate was about 10-20% in the carbon free medium, whereas the growth rate of 40-50% was shown in the medium (LOM1) to which the Chedamashima oligosaccharides were added (Table 1). One). In addition, the degree of acid production was similarly generated in the carbon free medium, but it was confirmed that the production in proportion to the growth rate in LOM1 medium (Table 2). Finally, as a result of TLC of the oligosaccharides, it was confirmed that the oligosaccharides were rapidly consumed after 8 hours of incubation time for Lactobacillus reuteri KCCM 40717 and Lactobacillus fermentum KCCM 40401. (FIGS. 6, 7 and 8). Thus, Chadashima oligosaccharides are expected to have a great effect on formal active functionality, showing the potential to be developed as formal functional biohealth materials.

Comparison of Relative Growth Rates of Bifidobacterium and Lactobacillus in RC or MRS Media Containing Glucose (Control), Laminarin Oligosaccharide Mixture (LOM1), or No Carbon Source Strain Relative Growth (%) Control LOM1 badge Carbon source free medium Bifidobacterium Infante Tees (B. infantis) KCCM 11207 100 52 8 Bifidobacterium adolescents KCCM 11206 100 47 11 L. fermentum KCCM 40401 100 45 19 L. reuteri KCCM 40717 100 42 26

Comparison of Acid Production of Bifidobacterium and Lactobacillus in RC or MRS Media with Glucose (Control), Laminarin Oligosaccharide Mixture (LOM1) or without Carbon Source Strain pH Control LOM1 badge Carbon source free medium Bifidobacterium Infante Tees (B. infantis) KCCM 11207 5.08 5.34 6.13 Bifidobacterium adolescents KCCM 11206 4.82 5.43 6.07 L. fermentum KCCM 40401 5.28 6.04 6.54 Lactobacillus rutheri KCCM 40717 5.06 6.17 6.52

As described above, the present invention refers to the culture supernatant of the Escherichia coli strain into which pLK108S (KFCC-11303) is inserted or the endo-beta-1,3-glucanase recovered therefrom. It is a method for producing oligosaccharides by adding the laminarin polysaccharide fraction purified from the enzyme to enzymatic reaction, and is very useful for the food and pharmaceutical industry because it has an excellent effect of producing oligosaccharides from Chedamashima in a short time with high efficiency.

Figure 1 shows the extraction process of the laminarin polysaccharide fraction.

2 is a gel filtration chromatogram of laminarine polysaccharide fraction.

Figure 3 is an HPLC chromatogram showing the molecular weight of the laminarine polysaccharide fraction (LP1).

FIG. 4 is a thin layer chromatogram (TL C) of true dashima derived laminarin hydrolyzed by endo-beta-1,3-glucanase derived from E. coli DH5α. to be.

FIG. 5 is an HPLC profile of the LP1 fraction from Chadashima hydrolyzed by endo-beta-1,3-glucanase.

6 is a TLC result showing the availability of oligosaccharides by L. reuteri KCCM 40717 as a control. Lane 1 represents the standard substances glucose (G1) and cellobiose (G2) as controls. Lane 2 is before incubation with 1% glucose, lane 3 after 8 hours of incubation, lane 4 after 12 hours of incubation and lane 5 after 24 hours of incubation.

FIG. 7 is a TLC result showing the oligosaccharide utilization of Laminarin Oligosaccharide Mixture 1 (LOM1) by L. reuteri KCCM 40717. Lane 1 represents the standard substances glucose (G1) and cellobiose (G2) as controls. Lane 2 is before incubation with 1% glucose, lane 3 after 8 hours of incubation, lane 4 after 12 hours of incubation and lane 5 after 24 hours of incubation.

FIG. 8 is a TLC result showing oligosaccharide utilization of Laminarin Oligosaccharide Mixture 1 (LOM1) by L. fermentum KCCM 40401. FIG. Lane 5 shows standard substance glucose (G1) and cellobiose (G2) as controls. Lane 4 is before incubation with 1% glucose, lane 3 after 8 hours of incubation, lane 2 after 12 hours of incubation, lane 1 after 24 hours.

Claims (4)

Preparation of oligosaccharides derived from Chedamasima, characterized in that oligosaccharides are prepared by adding the culture supernatant of Escherichia coli strain into which pLK108S (KFCC-11303) is inserted to laminarin isolated from Chamdashima. Way. Oligosaccharide was obtained by adding endo-beta-1,3-glucanase recovered from the culture supernatant of Escherichia coli strain into which pLK108S (KFCC-11303) was inserted, followed by enzymatic reaction. Method for producing a true shimashima derived oligosaccharide, characterized in that for preparing. The laminarin of claim 1, wherein Hydrothermal extraction of the dried kelp; Adding a 4-fold volume of ethanol to the extract to form a precipitate and centrifuging at 5000 rpm for 20 minutes (CLP); Performing superfiltration of the supernatant of the hydrothermally extracted crude extract using the 0.1-m ² membrane (LP); Filtering the solution obtained in the above step using a 100 kD membrane to remove the polymer; Concentrating the solution obtained in the above step using a 1 kD membrane and then lyophilizing to remove the salt of true kelp; Concentrating the crude polysaccharide 10-fold using hot water extraction and minitan-II and then gel filtration (LPI); And Method for producing a true shimashima oligosaccharide, characterized in that the total equivalent of the CLP, LP and LP1 fractions of the above step is measured through a sulfuric acid-phenol assay and purifying the fraction containing laminarin polysaccharides. The method for producing chadashima-derived oligosaccharides according to claim 1 or 2, wherein the oligosaccharides are laminaribiose or laminalipentaose.